L-Amino acids are used in human medicine, in the pharmaceutical industry, in the food industry and particularly in nutrition of animals.
L-amino acids such as, for example, L-lysine, are produced by fermentation of strains of the genus Corynebacterium, in particular Corynebacterium glutamicum. Because of its great economic importance, work is continually being done on improving the production methods. Improvements may relate to fermentation technology such as, e.g., stirring and supplying oxygen, or to the composition of the nutrient media, e.g., the sugar concentration during fermentation, or to the processing of the fermentation broth to a suitable product form by, e.g., drying and granulating the fermentation broth or ion exchange chromatography or may relate to the intrinsic performance properties of the microorganism itself.
The methods used for improving the performance properties of these microorganisms are those of mutagenesis, selection and screening of mutants. The strains obtained in this way are resistant to anti-metabolites or are auxotrophic for metabolites of regulatory importance, and produce L-amino acids. A known anti-metabolite is the L-lysine analogue S-(2-aminoethyl)-L-cysteine (see e.g., Tosaka, et al., Agricultural and Biological Chemistry 42(4), 745-752, (1978)).
Methods of recombinant DNA technology have likewise been used for a number of years for strain improvement of L-amino acid-producing strains of the genus Corynebacterium by modifying, i.e. enhancing or attenuating, individual L-amino acid biosynthesis genes and investigating the effect on L-amino acid production. Wendisch, et al. provide a review about amino acid production in C. glutamicum (World J Microbiol Biotechnol (2016) 32:105, 1-10).
Lee, et al. teach that the inactivation of a gene encoding a secretory protein results in an increase of L-lysine production in C. glutamicum (EP 3 141 597 A1). EP 3 144 383 A1 discloses that the inactivation of an intrinsic oxaloacetate decarboxylase enhances the L-lysine production in C. glutamicum. Möckel, et al. disclose that the attenuation of the poxB gene in C. glutamicum increases the production of L-lysine (EP 1 096 013 A2).
Ochrombel, et al. teach that the introduction of a glycine cleavage system into C. glutamicum increases the production of L-amino acids (EP 2 940 039 A1).
The nucleotide sequences of the chromosomes of various bacteria or strains resp. of the genus Corynebacterium and of the species Corynebacterium glutamicum, and their analysis have been disclosed. This information is available at publicly accessible databases and may be used for strain development purposes. One such data base is the GenBank data base of the NCBI (National Center for Biotechnology Information, U.S. National Library of Medicine 8600 Rockville Pike, Bethesda Md., 20894 USA).
During the annotation and submission procedure for a sequenced chromosome identified structures such as, e.g., genes or coding sequences are furnished with a unique identifier called locus_tag by the supplier of the information to the data base.
Corynebacterium glutamicum contains a gene in its chromosome which has an activity of conferring resistance to different drugs in Escherichia coli. 
Jäger, et al. (Journal of Bacteriology, 179(7), 2449-2451 (1997)) identified a gene termed cmr (corynebacterial multidrug resistance) in the chromosome of Corynebacterium glutamicum ATCC13032 which mediates resistance to several structurally unrelated antibiotics such as erythromycin, tetracycline, puromycin and bleomycin in Escherichia coli. This cmr gene confers a resistance phenotype only to Escherichia coli, but not to Corynebacterium glutamicum. Based on amino acid sequence analysis Jäger, et al. concluded that the gene encodes a hydrophobic protein with 12 potential transmembrane-spanning α-helical segments showing similarity to drug-H+ antiporters. Jäger, et al. further stated that the protein has a structure common for transport proteins belonging to the major facilitator family. However, the function of the activity of this protein in Corynebacterium glutamicum is still unknown. The nucleotide sequence of the cmr gene and the amino acid sequence of the encoded polypeptide are available under GenBank accession number U43535. The amino acid sequence of the encoded polypeptide is also shown under SEQ ID NO:1 of the sequence listing.
The nucleotide sequence of the Corynebacterium glutamicum ATCC13032 chromosome and its analysis was described by Ikeda and Nakagawa (Applied Microbiology and Biotechnology 62, 99-109(2003)) and in EP 1 108 790 A2. It is available at the NCBI under accession number NC_003450. Locus_tag NCgl2680 identifies a sequence coding for a “multidrug resistance protein”. EP 1 108 790 A2 also discloses nucleotide sequences coding for fragments of this multidrug resistance protein.
Nakagawa discloses under GenBank accession number NP_601971 the encoded amino acid sequence of a polypeptide defined as “multidrug resistance protein” of Corynebacterium glutamicum of ATCC13032. It is also shown under SEQ ID NO:2 of the sequence listing.
WO 2001/000804 A2 discloses various genes encoding stress, resistance and tolerance proteins (SRT proteins) from Corynebacterium glutamicum ATCC13032 and their use for the modulation of production of fine chemicals. Under identification code RXA01666 (page 59) and SEQ ID NO:233 and SEQ ID NO:234 a nucleotide sequence and the amino acid sequence of the encoded protein having the function of a multidrug resistance protein are disclosed. These sequences are also disclosed under GenBank accession number AX066329. It is stated that SRT proteins may be overexpressed (p37) or disrupted (p44). However, a specific application of said nucleotide sequence for the production of a specific fine chemical is not disclosed.
The amino acid sequences of entries U43535 and NP_601971 were compared and found to be identical over their full length. The two amino acid sequences were also found to be identical with the amino acid sequence of the multidrug resistance protein of ATCC13032 as disclosed in SEQ ID NO:234 of WO 2001/000804 A2 (see also GenBank accession number AX066329). Nishio, et al. disclose under GenBank accession number BAV24403 the encoded amino acid sequence of a protein defined as “permeases of the major facilitator superfamily” of a Corynebacterium glutamicum ssp. lactofermentum strain referred to as AJ1511. It is also shown under SEQ ID NO:3 of the sequence listing.
The nucleotide sequence of the chromosome of Corynebacterium glutamicum ATCC13869, a strain formerly referred to as Brevibacterium lactofermentum, and its analysis were disclosed by Chen, et al. at the NCBI under accession number NZ_CP016335. Locus_tag BBD29_RS13550 identifies a sequence coding for an “MFS transporter”. Chen, et al. disclose under GenBank accession number ANU34683 the encoded amino acid sequence of a protein defined as “multidrug transporter” of Corynebacterium glutamicum ATCC13869 (formerly referred to as Brevibacterium lactofermentum). It is also shown under SEQ ID NO:4 of the sequence listing.
The amino acid sequences of entries BAV24403 and ANU34683 were compared and found to be identical over the full length. When compared to the amino acid sequence of the corresponding polypeptide of ATCC13032 the identity was found to be 99.3%.
The nucleotide sequence of the Corynebacterium glutamicum R chromosome and its analysis were described by Yukawa, et al. (Microbiology 153(4):1042-1058 (2007)). It is available at the NCBI under accession number AP009044. Locus_tag_cgR_2674 identifies a sequence coding for a hypothetical protein comprising a region named “MFS”. “MFS” is the abbreviation for “Major Facilitator Superfamily”. Yukawa, et al. disclose under GenBank accession number BAF55689 the encoded amino acid sequence of a protein from Corynebacterium glutamicum R having a region named “MFS”. It is also shown under SEQ ID NO:5 of the sequence listing. Its identity to the corresponding amino acid sequence from ATCC13032 was found to be 98.9%.
The term “MFS” is the abbreviation for “Major Facilitator Superfamily”. According to the conserved domain database at the NCBI (see database entry cd06174) the term denotes a large and diverse group of secondary transporters that includes uniporters, symporters, and antiporters, which facilitate the transport across cytoplasmic or internal membranes of a variety of substrates including ions, sugar phosphates, drugs, neurotransmitters, nucleosides, amino acids, and peptides. Lv, et al. (Journal of Bacteriology 194(3), 742-743 (2012)) describe the sequencing and analysis of the chromosome of Corynebacterium glutamicum ATCC14067, a strain formerly referred to as Brevibacterium flavum. It is available at the NCBI under accession number AGQQ02000001 and AGQQ02000002. Locus_tag KIQ_001800 identifies a sequence coding for a “multidrug transporter”. Lv et al. disclose under GenBank accession number KEI24322 the encoded amino acid sequence of a protein from Corynebacterium glutamicum ATCC14067 (formerly referred to as Brevibacterium flavum) defined as a “multidrug transporter” having a region named “MFS”. It is also shown under SEQ ID NO:6 of the sequence listing. Its identity to the corresponding amino acid sequence from ATCC13032 was found to be 99.6%.
A summary of the findings is shown in table 1.
TABLE 1Comparison of the encoded amino acid sequences of Cmr polypeptides of various strainsof Corynebacterium glutamicum with the encoded amino acid sequence of the Cmr polypeptideof ATCC13032 (GenBank accession number NP_601971; see also SEQ ID NO: 2) by sequencealignment using the software program Clustal W (Larkin, et al., Clustal W and ClustalX version 2.0. In: Bioinformatics 23, 2947-2948 (2007)).CorynebacteriumglutamicumAccessionLength (number ofIdentical%SequenceStrainNumberamino acid residues)amino acidsIdentity(SEQ ID NO:)ATCC13032U43535459459100.0SEQ ID NO: 1AJ1511BAV2440345945699.3SEQ ID NO: 3ATCC13869ANU3468345945699.3SEQ ID NO: 4RBAF5568945945498.9SEQ ID NO: 5ATCC14067KEI2432245945799.6SEQ ID NO: 6
The amino acid sequence of the encoded Cmr polypeptide from ATCC 13032 shown in SEQ ID NO: 2 is also shown in SEQ ID NO:8.